1. Field of the Invention
The present invention generally relates to the field of microscopy and more particularly to techniques for exceeding diffraction-limit resolution in far-field microscopy by using nanoparticles co-doped with donor-acceptor dyes, laser pulses, and the method of fluorescence-resonance-energy-transfer-induced-emission-depletion (FRET-IED).
2. Description of the Related Art
The advancement in biological research has progressed very rapidly in the last couple of decades. One of the important tools helping this progress is microscopy that is used to obtain an enlarged view of an object so that it is possible to observe details that otherwise could not be observed. In order to view the precise location and the nature of the interactions between, for example, specific molecular species in live cells, a conventional far-field optical laser scanning microscope (LSM) has been used. LSM is used to observe tiny objects until it reaches a well-known limitation defined by Rayleigh criterion, which is also known as “diffraction-limit” is reached. As the light (i.e., laser) wavelength normally used in LSM is greater than about 400 nanometers (nm), the diffraction limit is approximately equal to 200 nm which is about one-half of the wavelength. Light waves emitted from a point source cannot be focused onto an infinite small spot by the objective lens of a microscope and, as a result, cannot distinguish two points at a distance less than the diffraction limit. Today, a microscope capable of resolution finer than 200 nm is needed in many biological research areas.
The electron microscope and short wavelengths (e.g., the X-ray) microscopes can achieve resolution less than 200 nm employing, respectively, electrons and X-ray as the scanning source. These sources have much shorter wavelengths as compared to normal laser light and, hence, carry high energies that can kill live cells during scanning. This defeats the purpose of observing live cells and thus is not a solution.
Another approach used to overcome the diffraction limit is the scanning near-field optical microscopy (SNOM). In SNOM, the light can be confined to a smaller size than the focal spot of the diffraction limit using a small aperture right above the object. However, this technique can only work within a very short decay distance from the small aperture due to the nature of the evanescent wave. As a result, SNOM is only good to scan the surface of the object. Similarly, an atomic force microscope (AFM), which can achieve a few nanometers of resolution, is limited to the observation of the object's surface. In other words, both SNOM and AFM are unable to achieve the desired three-dimensional images of, for example, live cells.
As a result, there is a need for breaking the diffraction limit using a conventional optical far-field microscope with one objective lens to observe live cells.